Hypoxia is a common feature of many solid tumors and is known to cause the resistance to chemotherapy and radiotherapy. Nitroreductase (NTR), a common biomarker of hypoxia, is an attractive target for the design of therapeutics and imaging agents targeting hypoxia. Under hypoxic condition, nitroreductase can reduce an aromatic nitro group conjugated to a drug or fluorophore to amine, which leads to release a free drug or fluorophore through the subsequent 1,6-rearrangment elimination. This type of 1,6-rearrangement elimination to release a drug or fluorophore has been widely used in the development of activity-based “turn on” fluorescent probes as well as targeted therapeutics such as antibody-drug conjugates (ADCs) and small molecule-drug conjugates (SMDCs). In this paper, novel “turn on” NTR-responsive fluorescent probes diversely linked to a 4-nitrobenzyl moiety via ether, carbonate, amine and carbamate linkages have been developed. The effect of variation in linkage on the release of fluorophore has been studied by the analysis of spectroscopic properties, temperature and pH stability, kinetics and concentration-dependency of the probes during NTR reaction. Fluorescent probes with an ether (10) or a carbamate (16) linkage showed biocompatible nature, high sensitivity (working below 1 μM of concentration) and strong fluorescence emission in the presence of NTR whereas they showed very low quantum yields in the absence of NTR. Finally, the probes (10, 16) were successfully applied to in vitro imaging of hypoxic cancer cells.